Quantum based effects of therapeutic nuclear magnetic resonance persistently reduce glycolysis

Summary Electromagnetic fields are known to induce the clock protein cryptochrome to modulate intracellular reactive oxygen species (ROS) via the quantum based radical pair mechanism (RPM) in mammalian cells. Recently, therapeutic Nuclear Magnetic Resonance (tNMR) was shown to alter protein levels of the circadian clock associated Hypoxia Inducible Factor-1α (HIF-1α) in a nonlinear dose response relationship. Using synchronized NIH3T3 cells, we show that tNMR under normoxia and hypoxia persistently modifies cellular metabolism. After normoxic tNMR treatment, glycolysis is reduced, as are lactate production, extracellular acidification rate, the ratio of ADP/ATP and cytosolic ROS, whereas mitochondrial and extracellular ROS, as well as cellular proliferation are increased. Remarkably, these effects are even more pronounced after hypoxic tNMR treatment, driving cellular metabolism to a reduced glycolysis while mitochondrial respiration is kept constant even during reoxygenation. Hence, we propose tNMR as a potential therapeutic tool in ischemia driven diseases like inflammation, infarct, stroke and cancer.


INTRODUCTION
The discovery of the core clock protein cryptochrome (CRY) as a receptor molecule for magnetic sensing in migrating animals gave rise to the question of a potential impact of man-made electromagnetic fields (EMFs) on the circadian clocks of somatic cells. [1][2][3][4] Although recent studies actually demonstrate that cellular clocks are affected by weak electromagnetic fields, knowledge on the underlying mechanisms or even on the physiological consequences is still scarce. Because the circadian system is intimately intertwined with the hypoxic signaling pathway, 5,6 the idea to study the associated hypoxic signaling pathway in response to EMFs, in our case a Radiofrequency (RF) EMF, was obvious. Only recently, we were able to show that the expression of Hypoxia-inducible factor-1 alpha (Hif-1a) indeed follows a nonlinear dose response relationship at the level of mRNA and protein in response to different treatment durations of therapeutic Nuclear Magnetic Resonance (tNMR, MBSTâ open system 350, MedTec Medizintechnik GmbH, Wetzlar, Germany, 0.4mT and 17 kHz). 4 We therefore decided to address the physiological consequences of the tNMR modulated Hif-1a levels observed and set out to investigate basic cellular metabolism such as glycolysis, mitochondrial respiration, cellular redox state and reactive oxygen species (ROS) signaling in response to tNMR. We performed the measurements after tNMR treatment under atmospheric oxygen saturation, which is actually hyperoxic for cells in culture, 7 and after tNMR treatment under acute hypoxia of 1% O 2 for six hours. Because hypoxic signaling is controlled by Hif-1a and rather well understood in mammalian cells, any potential impact of tNMR should be easier to observe and understand.
HIF-1a is a basic helix-loop-helix-PAS domain transcription factor responsible for oxygen dependent physiological cellular adaptations and oxygen distribution in cells, tissues and organisms. To form an active transcription factor HIF-1a heterodimerizes with its b subunit HIF-1b, the latter of which is constitutively expressed. The alpha subunit, in turn, though being constitutively expressed as well, undergoes constant degradation under normoxic conditions. This process of degradation under sufficient O 2 availability is initiated by the O 2 -dependent hydroxylation of proline residues 402 and/or 564 of the protein through prolyl hydroxylase domain protein 2 (PHD2). PDH2 consequently promotes the binding of the von Hippel-Lindau (VHL) protein, leading to subsequent ubiquitination of the protein and a final degradation by the 26S proteasome. In parallel, the asparagine residue 803 is hydroxylated by factor inhibiting HIF-1 (FIH-1), a process RESULTS tNMR affects the expression of Hif-1a at the mRNA and protein level under hypoxic conditions In our former publication 4 we reported the dose dependent nonlinear effects of tNMR on murine Hif-1a mRNA and protein expression in unsynchronized NIH3T3 cells. To explore eventual tNMR induced alterations of cellular metabolism, we now concentrated on the six-hour treatment, screening levels every 4 h, for two whole circadian cycles, using dexamethasone (Dex) synchronized NIH3T3 cells (Experimental setup shown in Figure S1). Under normoxic conditions we did not find significant differences in Hif-1a mRNA and protein expression ( Figures 1A and 1C) between sham and tNMR treated cells, which is in contrast to the data observed in unsynchronized cells. 4 The application of hypoxia (1% O 2 , 5% CO 2 and 94% N 2 ) for 6 h led to significant differences between normoxic control (sham normoxia) and hypoxic treated cells (sham hypoxia and tNMR hypoxia), Figures 1B and 1D). mRNA levels of Hif-1a were increased in hypoxic treated cells, and the increase was higher over the whole second day of sampling. During this period of time, between 24 and 48 h after the treatment, tNMR treated cells exhibited an even more pronounced and significant rise in mRNA levels compared to the solely hypoxia treated cells ( Figure 1B). At the level of protein, hypoxic treatment of cells led to a circadian oscillation of HIF-1a, as indicated by the red cosine wave fit to the data ( Figure 1D, p = 0.0145). Overall protein amounts of hypoxic treated sham cells appeared to be slightly decreased when compared to normoxic samples. This is not what one would normally expect, knowing that HIF-1a is stabilized under hypoxic conditions. We assume that the treatment with the cortisone derivative Dex might interfere with HIF-1a protein amounts, as reported previously. 22,23 The combined treatment of tNMR and hypoxia (tNMR hypoxia) led to significantly altered HIF-1a protein levels, namely a further overall reduction in protein amounts, but also a highly modified temporal expression of the protein ( Figure 1D). Differences were most pronounced between hypoxic sham and tNMR treated cells.

tNMR reduces lactate production and decreases cellular ADP levels under normoxic conditions
Because of the differences in Hif-1a expression we set out to investigate metabolites of cellular glycolysis in response to tNMR treatment under normoxic conditions, using the same experimental setup ( Figure S1). No differences were found in intracellular glucose and pyruvate levels (Figures 2A and 2B). Intracellular lactate was reduced during the first 24 h of sampling, which is one day after the tNMR treatment ( Figure 2C). Most prominent alterations were found in the intracellular ADP/ATP ratio, which was highly reduced in tNMR compared to sham treated cells ( Figure 2C). This reduction obviously was due to severely reduced intracellular ADP levels, while ATP stores were equal to control cells ( Figures 2E and 2F). No changes were found in the intracellular ratio of NAD + /NADH ( Figure 2G). In line with the reduced intracellular lactate levels ( Figure 2C), extracellular lactate concentrations ( Figure 2H)  iScience Article resulting in reduced overall lactate production ( Figure 2I). Hence, among the screened metabolites, only lactate and ADP were significantly affected by tNMR.
tNMR alters the metabolic signature after hypoxic treatment except for the NAD+/NADH ratio After hypoxic tNMR treatment intracellular glucose levels were reduced compared to normoxic cells ( Figure 3A). This commonly known hypoxia induced effect was significantly weakened after tNMR treatment under hypoxic conditions. Intracellular pyruvate, which was as well decreased in hypoxic control cells, appeared to be further decreased after tNMR under hypoxia ( Figure 3B). Astonishingly, the further decreased intracellular pyruvate levels did not result in an increased lactate production of tNMR treated cells under hypoxia. On the contrary, the combined treatment drove intracellular lactate levels towards those of normoxic cells, while solely hypoxia treated cells showed increased intracellular lactate levels as expected ( Figure 3C). In addition, tNMR under hypoxia further decreased the hypoxia induced decrease of the intracellular ADP/ATP ratio ( Figure 3D), by decreasing intracellular ATP levels less than those of ADP ( Figures 3E and 3F). After hypoxic conditions intracellular ATP stores were high, while tNMR lead to ATP stores residing in between normoxic and hypoxic cells. Intracellular ADP levels, in turn, were closer to those of normoxic cells. Conclusively, tNMR under hypoxic conditions led to relatively higher ATP versus ADP levels. No differences were found in the intracellular ratio of NAD + /NADH between hypoxic treatment and the combined treatment of tNMR and hypoxia, both being equally reduced in comparison to normoxic cells ( Figure 3G). As expected, extracellular lactate was highest in hypoxia treated cells, but reduced after the combined treatment tNMR and hypoxia ( Figure 3H), though still higher than that of normoxic control cells. Total production of lactate was highest in hypoxic cells, lower in hypoxic tNMR treated cells and lowest in normoxic control cells ( Figure 3I). In summary, tNMR treatment under hypoxic conditions significantly affected all measured metabolites in comparison to sham hypoxic cells, except for the intracellular ratio of NAD + /NADH. To characterize the impact of tNMR on the PPP, we measured the ratios of NADP + /NADPH and the activity of the PPP rate limiting enzyme Glucose-6-Phosphate-Dehydrogenase (G6PDH) after normoxic and hypoxic treatment ( Figure 4). Increased NADP + /NADPH ratios observed after the tNMR treatments indicate a relative decrease in the reduction equivalent NADPH under both oxygen tensions ( Figures 4A and 4B). Sham treated cells exhibited significantly reduced ratios after the hypoxic treatment. G6PDH activity was not altered in normoxic sham treated cells, but significantly reduced after hypoxic tNMR treatment at two specific time points ( Figures 4C and 4D). In summary, tNMR led to a rather reduced flux through the PPP immediately after the treatments, compared to sham treated cells. Interestingly, the decreased flux through the PPP after tNMR treatment occurred against the background of an increased cell proliferation, observable under both oxygen tensions ( Figures 4E and 4F). iScience Article generated (see Figure S2 for the experimental setup). tNMR led to a significant increase in extracellular ROS levels after normoxic and hypoxic treatment (Figures 5A and 5B) compared to sham cells, which produced less extracellular ROS after hypoxic incubation for six hours. Mitochondrial ROS was significantly increased after tNMR under normoxia ( Figure 5C), and the treatment under hypoxic conditions further enhanced the rise in mitochondrial ROS ( Figure 5D). No alterations were found in cytosolic ROS levels after normoxic tNMR treatment ( Figure 5E), whereas tNMR applied under hypoxia led to cytosolic ROS levels closer to those observed for normoxic sham treated cells than to those of hypoxic cells ( Figure 5F).

tNMR under normoxic conditions reduces the extracellular acidification rate (ECAR)
To further assess the metabolic phenotype of tNMR treated NIH3T3 cells we used a Seahorse Extracellular Flux Analyzer and respective mitochondrial and glycolysis stress test kits (Agilent) using the experimental setup shown in Figure S2. Normoxic tNMR treatment did not alter mitochondrial respiration (OCR) ( Figures 6A and 6B), but reduced the ECAR in tNMR treated cells ( Figure 6C). Though a trend towards a iScience Article reduced glycolysis was visible, the changes in the glycolytic parameters measured were not significant ( Figure 6D).

Hypoxic tNMR treatment reduces ECAR and throttles the hypoxia induced increase in glycolysis
Hypoxic sham treated cells responded to the reoxygenation with a significantly increased mitochondrial respiration (OCR), an increased basal and maximal respiration and a concomitant reduction in the spare respiratory capacity (in % of maximum respiration) ( Figures 7A and 7B). Mitochondrial respiration of  iScience Article hypoxic tNMR treated cells resided closer to those of normoxic sham treated cells, and also basal and maximal respiration were reduced compared to hypoxic sham cells. ECAR was significantly elevated in hypoxic sham cells, concomitant with a trend towards increased glycolysis and glycolytic capacity ( Figures 7C  and 7D), whereas tNMR treated cells under hypoxia exhibited ECAR's in between those of normoxic and hypoxic sham cells. Compared to hypoxic sham cells, the glycolytic reserve in % was higher in tNMR treated cells, which indicates a further capacity of tNMR treated cells to increase the glycolytic flux when needed.

DISCUSSION
The effects of weak EMF's on somatic cellular clocks, apart from the retinal CRY mediated magnetoreception in migrating animals, has been reported in several studies. [1][2][3][4] Knowledge on the underlying mechanisms is still scarce, although several groups increasingly focus on underlying quantum mechanical effects such as the RPM. 2,24,25,28 Even less known are any potential physiological consequences arising from the impact of EMF's on circadian clocks. Previously, we were able to show that tNMR did not only affect clock protein members in mammalian NIH3T3 cells, but also HIF-1a, 4 which is known to be tightly linked to the iScience Article circadian clock. 5,6 Because HIF-1a is known to regulate the adaption of cellular metabolism in response to altered oxygen tensions, we wanted to assess basic cellular metabolism after normoxic and hypoxic incubation with and without tNMR radiation, simulating therewith also a classical reoxygenation event. In a first experiment, we addressed HIF-1a mRNA and protein levels in Dex synchronized NIH3T3 cells after six hours of normoxic or hypoxic tNMR irradiation ( Figure 1, for experimental setup see Figure S1). Although unaltered under normoxic conditions, tNMR treatment substantially increased mHif-1a mRNA, surpassing levels of hypoxia sham treated cells during the second day of sampling. Counter-intuitively, protein levels of HIF-1a appeared to be reduced after hypoxia, and even more after the hypoxic tNMR treatment, which we partly assign to the HIF-1a protein reducing effect of the Dex treatment, according to, 23,29 and partly to the reoxygenation itself. Temporal expression of HIF-1a was as well significantly altered between hypoxic and hypoxic tNMR treated cells. Despite the observed decreased levels of HIF-1a protein, hypoxia treated cells elicited a canonical hypoxic response, indicated by decreased levels of glucose and pyruvate, decreased intracellular ratios of ADP/ATP and NAD + /NADH, whereas levels of intracellular ADP, ATP, intra-and extracellular lactate appeared to be increased ( Figure 3). Irradiation with tNMR under normoxia specifically reduced ADP and lactate concentrations (Figure 2), whereas the hypoxic irradiation resulted in a metabolic signature in between normoxic and hypoxic cells by reducing all measured metabolites in comparison to hypoxic sham cells, except for the NAD + /NADH ratio, which remained unaltered (Figure 3).
The apparent reduced glycolytic flux after hypoxic tNMR treatment led us to investigate the role of the PPP. Under both oxygen tensions, tNMR exposure appeared to increase the intracellular NADP + /NADPH ratios (Figure 4), indicating a decrease in NADPH, whereas sham treated hypoxic cells exhibited significantly reduced ratios compared to normoxic sham cells, hence an increase in the reducing equivalents NADPH. In addition, no activation of the PPP via the rate limiting enzyme G6PDH was found in any of iScience Article the treatment groups, quite the contrary, a slight reduction of enzyme activity at two specific time points was found in hypoxia tNMR cells. Accordingly, the results suggest that tNMR treated NIH3T3 cells did not need an increased flux through the PPP, at least after the treatment, neither for fighting unfavorable redox conditions through production of the reducing equivalent NADPH, nor for the delivery of ribose-5-phosphate for anabolic processes.
Cell proliferation rates, though, appeared to be significantly increased after tNMR treatment under both investigated oxygen tensions (Figures 4E and 4F). The influence of EMF's on cellular proliferation has been addressed repeatedly and was shown to depend on alterations in cellular ROS signaling, more specifically on mitochondrial ROS levels. 30 However, the reports are rather inconsistent. 3,24,31,32 Reasons for this seeming inconsistency of the correlation between EMF exposure and cell proliferation are the diverse types of EMF's used, 31 the localization of the measured ROS (extracellular, cytosolic, mitochondrial), the nonlinear dose response relationship 4,33,34 and most probably also the cell types investigated. Consistent with our data, increased proliferation rates after the treatment with RF EMF's were also reported by. 24,31 In accordance with these studies on RF EMF's we also found mitochondrial as well as extracellular ROS increased ( Figure 5). Interestingly, cytosolic ROS levels appeared to be reduced after the hypoxic tNMR treatment in comparison to hypoxic sham treated cells ( Figure 5F). The increased cytosolic ROS levels of the latter are most probably because of the increase in mitochondrial respiration (OCR) (Figures 7A and 7B), with which the cells were compensating for the reduced oxygen availability during the six-hour treatment of tNMR under hypoxia of 1%. Hence our experiment simulates also a classical ischemia reperfusion (IR) event, which is known to perturb redox balance iScience Article and to cause injury under pathophysiological conditions. 17,35,36 tNMR applied under hypoxic conditions appeared to reduce both during reoxygenation, mitochondrial respiration as well as cytosolic ROS levels.
Increased extracellular as well as mitochondrial ROS production after EMF exposure have been shown to be generated through the spin correlated RPM. This quantum based mechanism is an accepted model which explains the modulation of spin correlated radical pair states produced by semiquinone flavin (FADH) enzymes and O 2 -. Magnetic field alterations thereby lead to changes in the product ratio between singlet product yield (H 2 O 2 ) and triplet product yield (O 2 ,À ), which eventually affect the outcome of cellular ROS product ratios. The RPM was characterized in detail for the clock protein CRY, which meanwhile is an accepted magnetic receptor to enable compass orientation of migrating animals. 26,37 In addition, CRY was shown to be responsible for the intra-as well as extracellular ROS accumulation after exposure to a pulsating electromagnetic field of 1.8mT and 10 Hz, using murine mCry1/mCry2 double knockout cells 3 Apart from CRY, the RPM was also found to occur at the protein complexes of the mitochondrial electron transport chain, 25 and at several ROS production entities throughout the cells which depend on Flavindependent enzymes 38 , like the NADPH oxidase which transports electrons from NADPH across the plasma membrane to O 2 , resulting in the production of O 2 -. 2 Changes in the product yields of radical pairs are predicted for fields at frequencies corresponding to hyperfine couplings in the range between 1 to 100 MHz. 39 The field intensity of 0.4 mT in combination with a RF of 17 kHz used in our study to induce water proton nuclear magnetic resonance are clearly far below that range. However, we found elevated extracellular ROS levels, which from our current data set we can neither attribute to CRY nor to NADPH oxidase activity. The observed alterations in cytosolic and mitochondrial ROS signaling, as well as in mitochondrial respiration and glycolysis remarkably resemble the RPM based effects reported by. 25 The authors used primary human umbilical vein endothelial cells (HUVECs) and compared the effects of a single static magnetic field (MF) and a combined RF EMF on cellular bioenergetics. They found that H 2 O 2 and O 2 ,À product yields depended on the angle of the applied RF (parallel or perpendicular) and that either OCR or ECAR of the cells were affected, depending again on the angle of the applied field. Under both situations though, either OCR or ECAR appeared to increase. Beside the different cell types used, Usselman et al. 25 did not carry along a sham control, which means cells without any artificial MF exposure. Furthermore, our approach to study the effects of tNMR combined with two different oxygen tensions (normoxia and hypoxia of 1% O 2 ) adds additional complexity for comparison. tNMR under both, normoxic and hypoxic conditions appeared to reduce ECAR by throttling glycolysis (Figures 6 and 7), whereas OCR was kept constant under normoxia , and the reoxygenation induced rise in OCR of hypoxic cells was strongly diminished. Hence, the OCR of hypoxic tNMR treated cells during reoxygenation can be interpreted as ''kept as constant as possible'' in comparison to hypoxic sham cells.
Because there literally are no studies on the cell physiological effects of proton nuclear resonances we can only hypothesize which molecules or processes might be directly or indirectly affected by them. Only recently, the (re) orientation of water was demonstrated to control hyperfine electronic couplings in CRY. More specifically, the hydrogen bonding between the trytophans B and C in the CRY protein, which is necessary to form an electron-tunneling route, was demonstrated to be affected by the motions of the captured single water molecule and thus to depend on the local water solvation dynamics. 40 Apart from CRY, we assume that also mitochondrial flavoproteins, such as the Electron transfer flavoprotein or the Lipoamide dehydrogenase, 41,42 might be affected by proton resonances. This would also explain the rise in mitochondrial ROS production we observed in tNMR treated cells under hypoxic conditions ( Figure 5D).
Given that hypoxia prevails in cells and tissues of organisms and that pathophysiological reoxygenation events are known to be even more problematic than hypoxia itself after infarct and stroke, 17,35,36 the potential of tNMR to alter cellular metabolism even under or after low oxygen tensions is probably the most important finding of the present study. The obvious potential of tNMR to modulate cellular bioenergetics might also help to explain the recently reported effects of the treatment on the regeneration of primary rat dorsal root ganglion-derived Schwann cells in vitro 43 The role HIF-1a actually plays in the present settings, is still to question. HIF-1a is known to be directly regulated by ROS 44 and to regulate cellular and, in particular, mitochondrial metabolism itself. 45 In addition, HIF-1a is also known to be negatively regulated by CRY1. 46 Against this background the observed sensitivity of HIF-1a to changes in the external MF does not seem to be that exceptional. Even a quantum based ll OPEN ACCESS 10 iScience 25, 105536,  iScience Article direct regulation of HIF-1a through RF EMF's in general is conceivable. As already mentioned, protein levels of HIF-1a are regulated through hydroxylation of the proline residues 402 and/or 564 in an oxygen dependent manner. tNMR might directly affect these regulatory hydroxylation events of the protein through the proton resonance conditions induced. This idea has already been proposed for the DNA repair enzyme family AlkB by. 47 In addition, the stabilization of HIF-1a protein was shown to depend on the concentration of cellular FAD. 48 However, metabolic reprogramming as a therapeutic tool seems promising and has already repeatedly been suggested. 28,49,50 The increased HIF-1a driven glycolytic flux, which is commonly accompanied by increased lactate concentrations, is often a problem per se in pathophysiological conditions such as inflammation, infection (Covid-19), ischemic diseases as infarct and stroke, and also in tumor development and progression. The present study implicates that tNMR might have the potential to counteract the Warburg effect known from many cancer cells which are prone to glycolysis even under aerobic conditions. In this context, even very low doses of ionizing radiation commonly used for the treatment of tumors have been shown to increase glycolysis and lactate production, 51 which does not seem to be the case for tNMR at first sight. Hence, we strongly recommend to cautiously investigate tNMR as treatment option for pathophysiological conditions, in which a rewiring of basic cellular metabolism might be of advantage, given that no side effects of the treatment have been reported so far over the last two decades.

Limitations of the study
The dose response relationship between electromagnetic fields and biological matter is nonlinear, as mentioned above. This necessarily means that the results presented here only refer to the applied field intensity, frequency and the duration of 6 h. A longer or shorter duration of the treatment might therefore have a completely different outcome, as already demonstrated in. 4

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:   All requests for resources or reagents should be directed to and will be fulfilled by the lead contact author.

Data and code
All data reported in this paper will be shared by the lead contactupon request.
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

METHOD DETAILS
Experimental setup and sampling iScience Article